This invention relates to an implement containing oxidase, for analyzing body fluids, particularly body fluid components.
Analysis by enzyme reaction is suitable for the analysis of liquid samples because it provides specific detection under mild conditions. The oxidase reaction is widely used. With these methods, a sample material or its reaction product is oxidized by an oxidase. The hydrogen peroxide produced thereby, is reacted with a peroxidase to form a coloring matter, and the coloring matter is determined by colorimetry. Other detecting means such as fluorimetry, emission spectroscopy, and various types of electrodes are also available.
Well known methods of colorimetric analysis of hydrogen peroxide include the method using "Trinder's reagent" (Ann, Clin, Biochem, Vol. 6, page 24, 1969) and using an oxidizable chromogen such as o-anisidine, benzidine, o-tolidine, and tetramethylbenzidine. In these methods, hydrogen peroxide produced by the action of an oxidase is reacted with a peroxidase, is coupled with aminoantipyrine and a phenol by oxidation. The resultant coloring material is determined. Alternatively, the oxidizable chromogen is directly oxidized to form coloring material. This system has an advantage that the same detection means can be used with different types of oxidases. Thus, its application to various analysis items is being widely investigated. Oxidases essential to clinical examination includes glucose oxidase, urecase, cholesterol oxidase, glyco-3-phosphoric acid oxidase, choline oxidase, acyl-CoA-oxidase, sarcosine oxidase, various amino acid oxidases, bilirubin oxidase, lactose oxidase, pyruvic acid oxidase, galactose oxidase, and glycerol oxidase.
Heretofore, a solution containing one such oxidase, peroxidase, and a chromogen has been used for body fluid analysis in clinical examination.
Alternatively, reagents comprising the above analysis composition integrally mixed and dried (hereinafter called "solid reagent") have been widely used to answer the fundamental requirements of quick and simple analysis.
For example, a test film of plastic film applied with a detection system of a dispersion of this oxidase and peroxidase in polymer has been disclosed in U.S. Pat. No. 3,630,957. U.S. Pat. No. 3,992,158 describes a multi-layer test film comprising a liquid permeable and light transmittable substrate provided thereon with a reagent layer and developing layer. Modified multi-layer test film, include those which have an added a barrier layer as in U.S. Pat. No. 4,066,403 specification, with a registration layer and radiation blocking layer as in U.S. Pat. No. 4,144,306; and with a migration blocking layer as in U.S. Pat. No. 4,166,093. Most of these embodiments of solid reagent contain oxidase.
Deficient oxygen supply in these methods using oxidases can cause errors in measurements due to a reduced degree of coloring.
The oxygen requirement for oxidation in a solid reagent is furnished by dissolved oxygen present in the solid reagent and atmospheric oxygen dissolved in the reaction system.
However, as is obvious from the theoretical base shown below, sufficient reaction is not to be expected from the dissolved oxygen in the sample alone.
Solubility of air in pure water is 0.0167 ml/ml at 25.degree. C. under 1 atm. of which oxygen is 0.0057 ml/ml. (Chemical Handbook compiled by Chemical Society of Japan, Basic Chapter II, page 621, 1965, Maruzen). The dissolved oxygen 0.0057 ml/ml is calculated as 25 .mu.mol/dl, but because of other substances also dissolved in the sample, the actual quantity of oxygen is less than 25 .mu.mol/dl.
Components of normal blood serum include, for example, glucose 360-580 .mu.mol/dl, cholesterol 360-670 .mu.mol/dl, triglyceride 34-152 .mu.mol/dl, and uric acid 12-42 .mu.mol/dl. In abnormal cases, these values increase. Since the above solid reagent is generally applied to the sample undiluted or diluted several times at most, it is obvious that the dissolved oxygen in the sample alone cannot completely oxidize the substrate.
Therefore, should only the dissolved oxygen be used, sufficient oxidation will be impossible when the concentration of the sample is higher than the normal range, even for a substance of very low concentration such as uric acid in serum or even when the concentration is in normal range for other substances in serum.
Oxidase in the above mentioned multi-layer test film is contained in the reagent layer between the sample developing layer and support layer, and deficiency of oxygen is remarkable.
The time required for the quantity of oxygen, decreased through consumption, to recover to the specified level is much longer than the reaction time, since the oxygen is supplied to the solid reagent only after it is dissolved into the liquid sample drop from the atmosphere. This is fatal to expediting and simplifying the process.
Even the test film described in U.S. Pat. No. 3,603,957, comprising only a reagent layer formed on a substrate body, has as a drawback that the single reagent layer cannot exhibit its effect of direct contact of its surface with air, since the sample dropped on the reagent layer form a liquid intermediate layer between the reagent layer and air. Thus, only the dissolved oxygen in the reaction system present before the reaction and a small quantity of oxygen absorbed through the part of the reaction longer but not covered by the sample are used for the oxidation reaction.
In other words, even in the case of single reagent layer, oxygen required by the oxidase reaction is not fully supplied, leading to inaccurate analysis.
Japanese Laid Open Patent Specification No. 57-208,997 discloses a multi-layer test film containing the oxidase in the outmost porous developing layer to solve the drawback of deficient oxygen. The porous nature of the oxidase-containing layer provides a larger area of contact with atmospheric oxygen. This type of test film provides a larger effect relative to an analysis implement having only a reagent layer on the support.
Even with this test film, the quantity of oxygen is still not sufficient, and it has been ascertained by the present inventors that the problem still remains, particularly for a sample containing a high concentration of the compound in question.